The present invention relates to the field of molecular biology, more particularly nucleic acid hybridization, Holliday junction formation and branch migration. In one aspect, the invention provides methods and reagents for detecting the presence of a difference between two related nucleic acid sequences. In preferred embodiments of the invention, the difference is a mutation, such as a point mutation, deletion or insertion. Practical applications of the invention include, but are not limited to, genotyping, discovery and detection of single nucleotide polymorphisms, characterization and quantitation of polynucleotides, mutation rate detection, gene expression analysis. Furthermore, the invention is capable of distinguishing between homozygous and heterozygous genetic variation.
The tendency of nucleic acids to bind selectively and specifically to complementary nucleic acids has been exploited in the development of numerous nucleic acid hybridization techniques. Not only are such techniques useful for detecting complementarity and/or identity between nucleic acid sequences (e.g.: quantitating differential gene expression level such as Northern blots, Southern blots and gene expression chip/micro-arrays), but in some cases they are exploited to be used for detecting differences between related nucleic acid sequences.
Current allele-specific-hybridization-based genotyping technology suffers from poor accuracy due to low specificity. In particular, current genotyping technology based on polynucleotide hybridization often displays insufficient specificity to distinguish or identify single nucleotide polymorphisms. For instance, specific DNA strands/oligos containing one version of a specific SNP can hybridize not only to a perfectly matched complementary DNA but also to non-perfectly matched ones such as those contain a second version of the specific SNP. Althought the hybridization is stronger between two perfectly complementary DNA strands than that between two non-perfectly complementary DNA strands (including those that have either a single or multiple base-pair-mismatch between the two complementary strands), a single-base-pair difference is usually too small to render a high enough specificity for SNP scoring. In contrast, gene-expression chips and/or micro-arrays often have much better specificity/accuracy than SNP chips/micro-arrays due to the fact that the hybridization between specific cDNAs and their corresponding oligos/DNA fragments immobilized on the chip/micro-array is very specific. Such hybridization does not necessarily depend on a single-base pair difference between two nucleic acids. The method disclosed herein addresses the problem by combining highly specific allele-specific holiday structure formation with nucleic acid hybridization techniques (e.g.: gene chip/micro-array). As a result, SNP chips/micro-arrays can achieve the same high level of specificity/accuracy as gene-expression chips/micro-arrays.
In Panyutin IG et al""s 1993 paper (Panyutin IG, Hsieh P, Formation of a Single Base Mismatch Impedes Spontaneous DNA Branch Migration (1993) J. Mol. Biol., 230:413-24.), a single-stranded oligo that is completely (or partially) complementary to a specific part of single-stranded M13mp18 viral DNA anneals to the viral DNA and form a partial duplex with either 1 (or 2) tail(s) at each end. The partial duplex formed between the oligo and the M13mp18 viral DNA can then form a four-way Holliday-like structure with an invading partial duplex with either 1 (or 2) complementary tails. The four-way Holliday-like structure then undergoes branch migration in the direction away from the tail(s) (It can not branch migrate back towards the tail(s) due to energy barrier: breaking existing H-bonds without forming new ones). For Holliday structures formed between single-tailed partial duplexes, a single (or multiple) base pair difference between the duplex part of oligo/M13mp18 partial duplex and the duplex part of the invading partial duplex poses enough energy barrier (2 H-Bondsxe2x86x920 H-bond) to impede the branch-migration and prevent the release of the annealed oligo, regardless of the presence or absence of Mg++. For Holliday structure formed between double-tailed partial duplexes, a single base pair difference (substitution, deletion or insertion) between the duplex part of oligo/M13mp18 partial duplex and the duplex part of the invading partial duplex poses enough energy barrier to impede the branch-migration and prevent the release of the annealed oligo ONLY in the presence of Mg++.
One method that has been proposed for detecting differences between related nucleic acid sequences involves forming a complex comprising a Holliday junction between the related sequences. In this method, described in U.S. Pat. No. 6,013,439, each member of at least one pair of non-complementary strands within the complex is labeled. The two labels generate a signal that is dependent upon the labels being in close proximity to one another. If there is a difference in the related nucleic acid sequences, the Holliday junction is stabilized, thus positioning the labels within close proximity to one another and thereby generating a signal. If, on the other hand, no difference exists between the two sequences, the Holliday junction is not stabilized and the complex dissociates into duplexes, eliminating the close proximity between the two labels and attenuating the signal. A determination is made whether a stabilized complex is formed, the presence thereof indicating the existence of a difference between the related sequences.
One problem with the above-identified method of detecting differences between related nucleic acid sequences is that it normally requires the use of labeled PCR primers to generate the labeled nucleic acid strands required for detection of the Holliday junction complex. Particularly when the nucleic acid targeted for analysis is genomic DNA, the use of labeled primers can be problematic owing to the concentration of primer that must be used and the ensuing interference that occurs in the presence of high levels of labeled primers. This is a significant disadvantage, since one of the primary practical applications of the methodology is for the analysis of genomic DNA. Furthermore, the preparation and use of labeled nucleic acids is costly and inconvenient, so that it would be desirable to have available effective methods for determining sequence differences that do not require the use of labeled polynucleotides or primers.
The present invention addresses the problems associated with the use of labeled polynucleotides and primers by providing novel methods and reagents for the rapid and efficient identification of differences between related nucleic acid sequences. As such, the invention constitutes a highly desirable and practical addition to fields of endeavor including molecular biology and medicine.
Based on Panyutin IG and Hsieh P""s finding, we designed a genotyping method that allows multiplexing of genotyping assays (tens of thousands of SNPs/mutations can be assayed simultaneously in one assay reaction) and eliminate the requirement of individual PCR reactions. As a result, our method has the potential to dramatically reduce the cost and improve the throughput for genotyping.
In one aspect, the present invention provides methods for detecting the presence or absence of a difference between two related nucleic acid sequences. The methods achieve sensitivities great enough to detect the presence of any difference between the nucleic acids, even single nucleotide polymorphisms. In the methods, a target nucleic acid and a reference nucleic acid are contacted under conditions in which they are capable of forming a four-way nucleic acid complex with a branch structure that is capable of migration. Under the contact conditions, if the reference nucleic acid and target nucleic acid are identical, branch migration is capable of going to completion resulting in complete strand exchange. If the reference nucleic acid and target nucleic acid are different, branch migration does not go to completion, resulting in a stable four-way complex. Detection of the stable four-way complex identifies the presence of a difference between the nucleic acids. A stable four-way complex can be detected with molecules that specifically bind such complexes, by gel electrophoresis or by specific isolation of the stable four-way complex.
In one exemplary embodiment, the present invention provides a method for detecting the presence of a difference between two related nucleic acid sequences. The method involves forming a four-way complex comprising both of the nucleic acid sequences in duplex form, wherein the nucleic acids within the four-way complex are either not labeled or one of the four nucleic acid strands forming the four-way complex has a label, subjecting the four-way complex to conditions allowing branch migration to occur wherein, if a difference between the two related nucleic acid sequences is present, branch migration in said four-way complex ceases and the four-way complex continues to exist as a stable four-way complex; and wherein, if no difference between the two related nucleic acid sequences is present, branch migration in the four-way complex continues until complete strand exchange occurs and the four-way complex resolves into two duplex nucleic acids, subjecting said four-way complex or its resolved duplex products after branch migration to conditions allowing the specific binding of reagent(s) to said four-way complex and the binding of said reagent(s) to said four-way complex produces a detectable signal, and detecting the signal produced upon the specific binding of said reagent(s) to said four-way complex as the presence of said four-way complex, the signal thereof being related to the presence of said difference between said nucleic acid sequences and the failure to detect said signal thereof being related to the lack of difference between said nucleic acid sequences.
In another exemplary embodiment, the present invention provides a method for detecting the presence of a difference between two related nucleic acid sequences. The method involves forming a four-way complex comprising both of the nucleic acid sequences in duplex form, wherein the nucleic acids within the four-way complex are either not labeled or one or more of the four nucleic acid strands forming the four-way complex has a label, subjecting the four-way complex to conditions allowing branch migration to occur wherein, if a difference between the two related nucleic acid sequences is present, branch migration in said four-way complex ceases and the four-way complex continues to exist as a stable four-way complex; and wherein, if no difference between the two related nucleic acid sequences is present, branch migration in the four-way complex continues until complete strand exchange occurs and the four-way complex resolves into two duplex nucleic acids, subjecting said four-way complex or its resolved duplex products after branch migration to conditions allowing the separation of the four-way DNA complex (Holliday structure) from the duplexes DNA and the isolation of the four-way DNA complex. DNA fragments comprising the four-way complex can then be used as probes for SNP scoring through hybridization with oligos/DNA fragments immobilized on chips/microarrays.